Distribution and associations for antimicrobial resistance and antibiotic resistance genes of Escherichia coli from musk deer (Moschus berezovskii) in Sichuan, China

This study aimed to investigate the antimicrobial resistance (AMR), antibiotic resistance genes (ARGs) and integrons in 157 Escherichia coli (E. coli) strains isolated from feces of captive musk deer from 2 farms (Dujiang Yan and Barkam) in Sichuan province. Result showed that 91.72% (144/157) strains were resistant to at least one antimicrobial and 24.20% (38/157) strains were multi-drug resistant (MDR). The antibiotics that most E. coli strains were resistant to was sulfamethoxazole (85.99%), followed by ampicillin (26.11%) and tetracycline (24.84%). We further detected 13 ARGs in the 157 E. coli strains, of which blaTEM had the highest occurrence (91.72%), followed by aac(3’)-Iid (60.51%) and blaCTX-M (16.56%). Doxycycline, chloramphenicol, and ceftriaxone resistance were strongly correlated with the presence of tetB, floR and blaCTX-M, respectively. The strongest positive association among AMR phenotypes was ampicillin/cefuroxime sodium (OR, 828.000). The strongest positive association among 16 pairs of ARGs was sul1/floR (OR, 21.667). Nine pairs positive associations were observed between AMR phenotypes and corresponding resistance genes and the strongest association was observed for CHL/floR (OR, 301.167). Investigation of integrons revealed intl1 and intl2 genes were detected in 10.19% (16/157) and 1.27% (2/157) E. coli strains, respectively. Only one type of gene cassettes (drA17-aadA5) was detected in class 1 integron positive strains. Our data implied musk deer is a reservoir of ARGs and positive associations were common observed among E. coli strains carrying AMRs and ARGs.


Introduction
Musk deer is one of the first-class protected and endangered animals in China [1].In captive environment, intestinal disease is the main disease that threatens musk deer breeding [2,3].
Antimicrobial resistance (AMR) of E. coli has become a global challenge due to the wide usage of antibiotics in clinic [4].In particular, E. coli isolates that were resistant to at least three or more categories of antibiotics are considered as multi-drug resistant (MDR) [5], which increased the difficulty of treating animal bacterial diseases and also a potential risk to public health.
Antibiotic resistance gene (AGR) was considered to be the main factor of antimicrobial resistance [6] and become a new contaminant for environmental pollution [7].ARGs are usually carried by integrons, transposons and plasmids to mediate the horizontal transmission of AMR [8].Integrons can capture, convert and adapt resistance gene cassettes into functionally expressed genes via self-efficient gene expression system [9].Integron usually links to plasmid, which can transfer ARGs among various bacterial species [10].
E. coli is usually considered as an indicator organism to AMR [6,11,12].Though AMR associated with ARGs have been reported in E. coli from different animals (honeybees, poultry and pigs) [13][14][15], antimicrobial resistance of E. coli in musk deer remains unknown.This study aimed to examine the antimicrobial resistance phenotype, antimicrobial resistance genes and integron in E. coli to better understand the association among AMR phenotypes and ARGs in musk deer.

Sample collection
From Sep to Oct 2020, fresh fecal samples were collected from 157 clinically healthy musk deer in Dujiang Yan (n = 101) and Barkam (n = 56) (S1 Table ).Each sample was collected from an individual.Fresh fecal specimens (approximately 10 g) from each musk deer were collected immediately by feeders after defecation on the ground and then quickly transferred into individual 50-mL plastic containers.After collection, samples were stored at 4˚C in a cooler and transported to the laboratory for bacterial isolation within 12 h.
This study was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number DYY-2020103018.Prior to the collection of fecal specimens from captive musk deer, permission was obtained from Sichuan Institute of Musk Deer Breeding in Sichuan, China.

Isolation and enumeration of E. coli
Fecal samples were pre-enriched in buffered peptone water (Oxoid, UK) and incubated for 4-6 h at 37˚C.All isolates were presumptively identified by Gram staining, MacConkey (Solarbio, BeiJing), and then confirmed by eosin methylene blue agar growth (Chromagar, France) (Solarbio, BeiJing), and then confirmed by eosin methylene blue agar growth (Chromagar, France) [16,17] and biochemically identified by API 20E system (BioMerieux, France).The 16SrRNA (F: AGAGTTTGATCCTGGCTCAG, R: GGTTACCTTGTTACGACTT) of all strains were further amplified to confirm the isolates are E. coli.

DNA extraction and screening for antimicrobial resistance genes and integrons
Total E. coli genomic DNA was extracted by kit according the manufacturer (Tiangen Biotech, Beijing, China).Extracted DNA was stored at -20˚C.Thirteen different resistance genes and intl 1, intl 2 and intl 3 gene amplified by polymerase chain reaction (PCR).Subsequently, strains harboring intl 1 or intl 2 genes were elaborated for the amplification of variable regions (VRs).The PCR products were separated by gel electrophoresis in a 1.5% agarosegel stained with GoldView™ (Sangon Biotech, Shanghai, China), visualized under ultraviolet light and photographed using a gel documentation system (Bio-Rad, Hercules, USA).Primers and detailed conditions for PCR are described in S2 Table .The PCR reaction mixture was prepared with a composition of 12.5 μL of master mix (Origin, India), 2 μL template DNA, 1 μL primers (forward and reverse) and the volume raised to 25 μL with the water free from nuclease.

Sequence and data analysis
All positive PCR products were directly sequenced by BGI (Beijing, China) in both directions.Sequences were analyzed online using BLAST (http://blast.ncbi.nlm.nih.gov).P-values < 0.05 were considered to be statistically significant.The association between AMR phenotypes and the ARG was calculated, and the association was considered significant at a P-value of < 0.05 and was reported as an odds ratio (OR) with 95% confidence intervals (CI).An OR of > 1 was considered as positive association or the increasing probability of the cooccurrence of the genotype or phenotype, while an OR of < 1 was considered as negative association or the decreasing probability of the cooccurrence of the genotype or phenotype.All analyses were conducted using the SPSS 27 software (StataCorp Lp, College Station, TX, USA).

Antimicrobial susceptibility testing for 157 E. coli
The distribution of antibiotic resistant in 157 E. coli was summarized in S1  rate of antibiotic resistance and showed no significant difference (P > 0.05).Significant differences in resistance to TE, KZ, CXM, SAM and CIP were observed from Barkam and Dujiang Yan (P < 0.05).

Distribution of 13 antibiotic resistance genes (ARGs) for 157 E. coli
As S1 The details of ARGs carrying rate for E. coli strains from captive musk deer in two farms were shown in Tables 3 and 4, Fig 1.Among strains that carried ARGs, 94 (93.07%, 94/101) strains were isolated from Dujiang Yan and 55 (98.21%, 55/56) strains were isolated from Barkam.The most prevalent ARGs pattern was bla TEM /aac(3')-IId in Dujiang Yan (29.70%, 30/ 101) and in Barkam (23.21%, 13/56), respectively.The ARGs carrying rate in Barkam was 98.21%, higher than that in Dujiang Yan (93.07%).In addition, bla SHV , catA1 and qnrA were not detected in Dujiang Yan, bla SHV and catA1 were not detected in Barkam.Significant difference in bla CTX-M , floR, tetA, sul2, aac(6')-Ib were observed between strains from Barkam and Dujiang Yan (P < 0.05).A: Eighty-six E. coli strains were resistant to RL and the bla TEM was the most prevalent genes among all tested genes in Dujiang Yan.B: The results were similar in Dujiang Yan, the most prevalent AMR was RL and bla TEM was the most prevalent genes among all tested genes in Barkam. https://doi.org/10.1371/journal.pone.0289028.g001

Discussion
To better understand the antibiotic resistance of E. coli from captive musk deer in Sichuan, China, 157 E. coli strains isolated were detected for the occurrence of AMRs, ARGs, integrons and gene cassettes.Among the 157 E. coli strains, 144 strains were resistant to at least one antibiotic (91.72%, 144/157), which is lower than data from hybrid deer as reported in another study (100.00%,51/51) [23].Meanwhile, 38 isolates in our study were found to be multi-drug resistant (MDR) and the rate of MDR (24.2%, 38/144) is higher than E. coli strains isolated from hybrid deer (3.30%, 1/30) [24].However, the rate of MDR in our result is lower than the rate in captive giant pandas (100.00%,89/89) and non-human primates (49.65%, 494/995) [16,    Odds ratio (OR) for significant associations between AMRs (95% confidence interval in parenthesis); Na, no results available (or) could not be calculated because none of the isolates carried one of the combinations of AMR or one of the values of antibiotic was a constant or zero);-indicates no significant associations (P > 0.05); NS, no statistics were determined for the same AMR.https://doi.org/10.1371/journal.pone.0289028.t00525], which showed MDR strains from musk deer is not serious compared with giant pandas and non-human primates.Our results showed E. coli from musk deer were mostly resistant to RL (85.99%),AMP (26.11%) and TE (24.84%) and the antibiotic resistance rates are lower than the corresponding resistance rate of E. coli from deer (RL, AMP, TE were 90.63%, 90.63% and 87.50%, respectively) reported in Bangladesh Nature Reserve [26].This phenomenon may relate to different antibiotic used in the two regions [27,28].However, the resistance rate against TE (24.84%),KZ (22.93%),ATM (14.65%),CHL (10.19%),CAZ (8.92%) and CN (6.37%) were higher than the rate previously reported from captive musk deer in Sichuan, China (TE, 20.34%; KZ, 8.47%, ATM, 1.69%; CHL, 6.78%; CAZ, 1.69% and CN, 5.08%) [29].This implied the antimicrobial resistance rate of E. coli in musk deer has increased [30,31].It is worth noting that our results showed that 85.53% of E. coli are resistant to RL, but two farms have not used any sulfonamides in recent years.This also suggests that there are more complex mechanisms for the generation of bacterial drug resistance in addition to antibiotic pressure [32,33].The rate of MDR from Barkam is higher than that from Dujiang Yan, indicated antibiotic resistant in Barkam is more severe and more caution should be exercised when choosing Odds ratio (OR) for significant associations between resistance genes (95% confidence interval in parenthesis); Na, no results available (or) could not be calculated because none of the isolates carried one of the combinations of resistance genes or one of the value of genes was a constant or zero);-indicates no significant associations (P > 0.05); NS, no statistics were determined for the same gene.
https://doi.org/10.1371/journal.pone.0289028.t006antibiotics.The antibiotic resistance rates of Barkam to TE, KZ, CXM, SAM and CIP are significantly higher than that from Dujiang Yan, which may relate to different living environments [34,35].The expression of antibiotic resistance genes is an important mechanism of antimicrobial resistance [36].In our study, 11 ARGs were detected and the most prevalent ARG was bla TEM (91.72%), the occurrence of which is higher than that from another study on musk deer (30.51%) [29].However, the detection rate of another ESBL-encoding gene bla CTX-M (16.56%) in our present study is lower than the previous result (35.59%) [29].As important ESBLencoding genes, bla TEM and bla CTX-M were both detected in E. coli implied feces excretion from musk deer posed a potential harm to environmental safety [37].The prevalence of tetB (7.01%) and sul3 (10.83%) from musk deer in our study were higher than the prevalence previously reported from other deer in Northeastern China [38].Moreover, aac(3')-IId (60.51%), aac(6')-Ib (8.28%) and qnrA (1.27%) genes were firstly detected in E. coli from musk deer.The above results suggest the carried rate of ARGs in musk deer are increasing.ARGs carried in clinically healthy animal (non-human primates, giant pandas and waterfowl birds) pose a potential threat to the surrounding environment [16,39,40].Our results showed that musk deer carried 11 kinds of ARGs and the surrounding environment was a potential threat by feces of musk deer, suggesting that the feces should be disinfected to reduce the environmental pollution caused by antibiotic-resistant E. coli.High prevalence of ARGs implied that E. coli from musk deer is a reservoir of ARGs, and continuously monitoring the occurrence rate of ARGs in E. coli from musk deer is needed in future.
Previous studies have shown that an association exist between AMR and ARGs in E. coli [41][42][43].Our result showed only positive associations (OR > 1) were observed between ARGs and AMRs, no negative associations were observed.This is similar to the another report, where only positive associations between AMRs and ARGs in Urinary tract patient were detected [44].Our study showed the AMR-ARG pair with the strongest positive association was CHL/floR (OR, 301.167; 95% CI, 46.078-1968.425).This is consistent with the results of E. coli from pigs, since chloramphenicol is positively associated with the corresponding AGRs [45].Although 9 pairs of AMRs and ARGs showed positive associations in our result, the antibiotic resistance phenotypes did not completely match the ARGs, which is similar to the findings from previous studies [46][47][48].In general, the antibiotic resistant phenotype of bacteria is determined by its ARGs [49][50][51].In our study, antibiotic resistant phenotype was not completely consistent to the ARGs detected.The reason may relate to the expression level of ARGs, which may not be high enough to exert drug resistance [52].Moreover, our present study using PCR to detect ARGs, not using whole gene sequencing, which may underestimate the detection rate of ARGs.Previous studies showed integron plays a vital role in the dissemination of ARGs [30,53,54).In our results, class 1 and class 2 integrons were all detected from musk deer, while class 3 integron was not detected.Class 1 integron (10.19%) was more prevalent compared with class 2 integron (1.27%), which is similar to previous reports [55][56][57].Gene cassette that harbored in integrons-positive isolates is an important media to spread ARGs to the environment [58,59].The distribution rate of integron (10.82%) from musk deer in our study is lower than that in other captive animals, such as little penguins (44.70%),Australian sea lion (50.00%) and giant panda (35.71%) [56,[60][61][62].Our result showed only dfrA17-aadA5 gene cassettes arrays were detected in class 1 integron-positive isolates.Trimethoprim resistance gene (dfrA17) and aminoglycoside resistance gene (aadA5) was observed for the first time in musk deer in our present study.The type of integron gene cassettes carried by E. coli from forest musk deer are lower than that from other animals, such as giant panda (6 types), yak (11 types) and nonhuman primates (18 types) [16,25,63).The results showed that there were a few kinds of integrons and gene cassettes carried by intestinal E. coli from forest musk deer.Positive results of class 1 and class 2 integrons detected in E. coli from forest musk deer implied drug resistance and horizontal transfer of ARGs cannot be ignored.

Conclusion
This is the first study detected the prevalence of antimicrobial resistance, antibiotic resistance genes and integron genes cassettes in E. coli obtained from fecal of musk deer.Positive associations were more common observed among E. coli strains carrying antibiotic resistance genes and antimicrobial resistance.Our results implied musk deer might act as an important reservoir of ARGs and integrons, which may spread into the natural environment and accompanying animals around musk deer.

Fig 1 .
Fig 1.Heat map demonstrating the distribution of ARGs and AMRs in E. coli isolates from musk deer in Sichuan, China.The color scale on the right of figure showed the total number of E. coli isolates, in each of which carried a pair of antibiotic resistance genes and antibiotic corresponding to abscissa and ordinate, blue indicating the number is low, white indicating intermediate value, and red indicating the number is high.The number of positive isolates were also listed in the figure.A: Eighty-six E. coli strains were resistant to RL and the bla TEM was the most prevalent genes among all tested genes in Dujiang Yan.B: The results were similar in Dujiang Yan, the most prevalent AMR was RL and bla TEM was the most prevalent genes among all tested genes in Barkam.
AMRs with a significant association (P < 0.05) are shown.